PROJECT SUMMARY Inherited retinal degenerations, including retinitis pigmentosa (RP), originate from mutations in nearly 200 genes affecting photoreceptor cells (rods in the case of RP). Therapeutic options for treating these diseases are limited. One possible approach to treat these conditions is to study the common pathobiological mechanisms downstream of multiple mutations. For example, many mutations linked to retinal degenerations cause photoreceptor proteins to fold poorly and to be targeted to the proteasome for degradation. This suggests a connection between altered cellular proteostasis (i.e. the balance between protein synthesis and degradation) and photoreceptor degeneration. So far, any mechanistic understanding of this connection is still in its infancy. The experiments in this proposal will evaluate the function of the ubiquitin-proteasome system (UPS), the cellular machinery that selectively degrades proteins, in the context of two mouse models of retinal degeneration linked to production of misfolded proteins. Protein degradation by the UPS is typically initiated when a protein is marked for degradation by ubiquitination, which enables the proteasome to recognize and degrade the protein. Many proteins also require processing by molecular complexes involving the chaperone P97 before they can be recognized by proteasomes. Recent work in multiple mouse models of retinal degeneration demonstrated that mutant rods suffer from insufficient UPS capacity prior to the onset of cell death. However, the specific UPS component that is limiting protein degradation in these models remains unknown. The goal of this proposal is to identify this limiting component, which will pave the way for the development of pharmacologic and gene therapy approaches for treatment of inherited retinal degenerations. Aim 1 of this proposal seeks to determine whether UPS-mediated protein degradation in mutant rods is limited by P97. This will be accomplished by measuring the accumulation in mutant rods of a P97-independent reporter of UPS function. Aim 2 will investigate the possibility that the UPS is limited is at the level of ubiquitination by comparing the rate of UbG76VGFP ubiquitination between WT and mutant mice treated with P97 and proteasome inhibitor. In Aim 3 the identified limiting UPS component will be overexpressed in mutant rods, and resulting UPS activity, rod degeneration, and rod function will be evaluated. The results of these Aims will identify the critical component of the UPS whose capacity is overwhelmed prior to rod death. The proposed work represents the first systematic examination of the UPS in the context of ongoing photoreceptor degeneration and will advance the understanding of the factors underlying the pathology of this complex eye disease.